Elsevier

Immunology Letters

Volume 84, Issue 3, 3 December 2002, Pages 217-221
Immunology Letters

Endomorphin-1 alters interleukin-8 secretion in Caco-2 cells via a receptor mediated process

https://doi.org/10.1016/S0165-2478(02)00198-0Get rights and content

Abstract

Administration of opioids that bind to the classical μ opioid receptor has been shown to lead to unintended alterations in immune function. Traditionally, altered immune function has been investigated with circulating immune cells. Effects of μ agonists on intestinal epithelial immune function have not been described. Since the oral route of administration is frequently employed with opiates, we determined if the μ receptor specific agonist endomorphin-1 altered interleukin-8 (IL-8) production by Caco-2 cells. Using RT-PCR and immunocytochemistry, Caco-2 cells were found to constitutively express (mu) μ opioid receptors. Activation of the μ receptor by endomorphin-1 (1 and 10 μM) resulted in significant increases in IL-8 when Caco-2 cells were stimulated with IL-1β. Increased IL-8 secretion due to endomorphin-1 could be blocked by pre-incubating cells with the μ receptor antagonist, β-funaltrexamine. These results indicate that the intestinal epithelial IL-8 response can be altered by a μ opioid receptor mediated mechanism.

Introduction

It is well known that opiate abuse adversely affects immune function. Altered immune function is postulated to be mediated through opioid receptors located in the central nervous system as well as those located on peripheral immune cells including T-lymphocytes, macrophages, and natural killer cells [1]. Many of the characterized effects are the result of agonist binding to the (mu) μ opioid receptor. While considerable research indicate that chronic use of μ opioids such as morphine result in reduced proliferative responses in T-lymphocytes [2] and altered cytokine release from macrophages [3], little is known concerning the affects of μ opioids on intestinal immune function.

The intestinal epithelium is integral to the body's interaction with intestinal bacteria, food antigens, and in the pathogenesis of disorders such as inflammatory bowel disease. One mechanism by which intestinal epithelial cells (IEC) participate in mucosal immunity is the release of chemokines. Of the chemokines produced by IEC, interleukin-8 (IL-8) has been identified as a key mediator of epithelial immune responses. Production of IL-8 by IEC serves as an important signal, which influences the activation, growth, differentiation, and migration of numerous target cells located in the intestine. Therefore, if μ opioids alter IL-8 secretion by IEC, this could have deleterious effects on the local inflammatory response. To test this hypothesis, Caco-2 cells were treated with the endogenous μ tetrapeptide endomophin-1 (Tyr–Pro–Trp–Phe–NH2). Unlike other μ ligands such as morphine, endomorphin-1 is highly selective for the μ opioid receptor and does not bind either δ or κ receptors [4]. In the present study, the activation of μ opioid receptors on Caco-2 cells by endomorphin-1 increased IL-8 secretion in the presence of an inflammatory stimulus. This effect was reversible using a μ opioid receptor selective antagonist.

Section snippets

Cell culture

The human IEC line Caco-2 was obtained from American Type Culture Collection (Rockville, MD) and incubated in a humidified atmosphere of 5% CO2 at 37 °C. Cells were maintained in Dulbecco's modified Eagle's media supplemented with 25 mM glucose (Mediatech, Herndon, VA), 10% fetal bovine serum, 1% non-essential amino acids, 2 mM l-glutamine, 1 mM sodium-pyruvate, and penicillin/streptomycin (100 U/100 μg/ml) (Sigma, St. Louis, MO). Media was changed every other day. Near confluent monolayers were

Caco-2 cells constitutively express μ opioid receptors

In order to conclude the effects of endomorphin-1 on IL-8 secretion were receptor mediated, it was first necessary to determine if Caco-2 cells express μ opioid receptors. As shown in Fig. 1, Caco-2 cells constitutively express μ opioid receptors as detected with RT-PCR. These findings were confirmed by immunocytochemistry (Fig. 2). Mu opioid receptors were visualized throughout the apical surface of the cell. Using an optical sectioning technique, it was determined that μ opioid receptors were

Discussion

Activation of the μ opioid receptor on circulating immune cells has predominantly been associated with diminished chemokine responses [6], [7]. However, there is evidence for augmented chemokine effects. Wetzel and colleagues found that the μ selective opioid agonist (DAMGO) increased the expression of the proinflammatory chemokines, monocyte chemoattractant protein-1, RANTES, and IFN-γ-inducible protein (IP)-10 in human peripheral blood mononuclear cells [8]. However, effects of μ opioid

Acknowledgements

The authors are grateful for the assistance of Jessica M. Buck. Ms. Buck assisted with the IL-8 ELISA.

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